Antifungal activity and mechanism of the essential oils from (Litsea cubeba), Melissa (Melissa ofcinalis), Palmarosa ( martini) and Verbena (Verbena ofcinalis) and their major active constituents against Trametes hirsuta and Laetiporus sulphureus

Yongjian Xie (  [email protected] ) Zhejiang A & F University https://orcid.org/0000-0001-8225-7720 Xi Yang Zhejiang A&F University Hui Han Zhejiang A&F University Zhilin Zhang Hubei Engineering University Dayu Zhang Zhejiang A&F University

Research Article

Keywords: Antifungal activity, Essential oils, Litsea cubeba, Geranial, Membrane damage

Posted Date: August 13th, 2021

DOI: https://doi.org/10.21203/rs.3.rs-680348/v1

License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License

Page 1/24 Abstract

Antifungal activities of 37 essential oils (EOs) against two wood-decaying fungi, Trametes hirsuta and Laetiporus sulphureus were screened in vitro, and investigated the underlying mechanism. Of the 37 EOs, litsea (Litsea cubeba), melissa (Melissa ofcinalis), palmarosa (Cymbopogon martini), and verbena (Verbena ofcinalis) demonstrated strong antifungal activity, in which litsea oil exhibited the strongest antifungal property against T. hirsuta and L. sulphureus, with IC50 values of 72.3 and 40.2 µg/ml, respectively. The compositions of litsea, melissa, palmarosa, and verbena EOs were analyzed using a gas chromatography-mass spectrometry method and demonstrated geranial, geraniol, neral, and citral as their major active constituents. Among of them geranial exhibited the strongest antifungal activity against T. hirsuta and L. sulphureus, with IC50 values of 56.6 and 33.3 µg/ml, respectively. These EOs and their major active constituents increased the plasma membrane permeability of T. hirsuta and L. sulphureus, resulting in the leakage of nucleic acid, protein, and soluble sugar. Results indicate that the EOs of litsea, melissa, palmarosa, and verbena and its major constituents inhibited T. hirsuta and L. sulphureus growth by targeting its plasma membrane.

1. Introduction

The biodegradation of lignocellulosic materials occurs by beetles, fungi, marine borers, and termites; among these, fungi are recognized to cause the greatest fnancial loss of wooden products (Wu et al. 2012). Decay fungi, molds, and stain fungi are universally recognized as major wood-degradation fungi (Bakar et al. 2013). In general, traditional wood preservatives are ammoniacal copper quaternary (ACQ), chromated copper arsenates (CCAs), copper azole (CA), and copper (II) dimethyldithiocarbamate (CDDC), which signifcantly affect human health and the environment (Chen et al. 2014). Therefore, there is an urgent need to research and explore more eco-friendly, convenient, and highly effective benign wood preservatives for lignocellulosic materials.

In recent years, many natural products that are non-residual, biodegradable, and environmentally friendly have been shown to be excellent potential alternatives for preserving the wood industry (Xie et al. 2017a). Studies have shown that plant essential oils (EOs) from Calocedrus fromosana, Cryptomeria japonica, Cinnamomum osmophloem, Ci. zeylanicum, Cymbopogon citratus, Cunninghamia konishii, Eucalyptus camaldulensis, Eugenia caryophyllata, Machilus philippinensis, Origanum vulgare, Pelargonium graveolens, and Thymus vulgaris have antifungal properties (Cheng et al. 2005, 2006, 2011; Ho et al. 2010; Xie et al. 2015, 2017a; Salem et al. 2016). As is well known, the strong antifungal activity of EOs against wood decaying fungi were contributed to a rich of monoterpenes, sesquiterpenes and phenylpropanoids (Cheng et al. 2012; Zhang et al. 2016a). Most EOs and their compounds destroy the integrity of fungal cell membranes, resulted in the outfow of intracellular components and cell death (Kalily et al. 2016; Zhang et al. 2016b; Zhou et al. 2017; Souza et al. 2020; Yan et al. 2020).

Research on EOs as biological agents for protecting wood and prolonging their application life and as an alternative to chemical wood preservatives is becoming increasingly a necessity. For this reason, this

Page 2/24 study examined the antifungal activity of 37 EOs against wood-decay fungi. We also analyzed the chemical composition of EOs with the strongest antifungal activity by gas chromatography-mass spectrometry. In addition, we evaluated the antifungal activity of the major active constituents in the selected EOs and elucidated the relationship between the active constituents and their antifungal properties. Finally, we investigated the changes in plasma membrane permeability of T. hirsuta and L. sulphureus caused by selected EOs.

2. Materials And Methods 2.1. Wood decay fungi

Trametes hirsuta (CFCC 84683) and Laetiporus sulphureus (CFCC 86368) were procured from China Forestry Culture Collection Center. 2.2. Essential oils and chemicals

The antifungal activities of 37 EOs against wood rot fungi in vitro were screened (Table 1). Litsea cubeba and Verbena ofcinalis were procured from Rihua Chemical Co. Ltd. (Guangzhou, China). The other EOs was purchased from Huien International Business Co. Ltd. (Shanghai, China). Geraniol, neral, citral, and geranial were purchased from Sigma-Aldrich (China).

Page 3/24 Table 1 List of plant essential oils tested for wood-decay fungi oil source of plant family name part origin

Chamomile Anthemis nobilis Compositae Flower France Wormwood Artemisia argyi Compositae China Cypress Cupressus sempervirens Cupressaceae Leaves France Juniper berry Juniperus communis Cupressaceae Fruit France Palmarosa Cymbopogon martini Gramineae Leaves Brazil Citronella Cymbopogon winterianus Gramineae Leaves Java Vetiver Vetiveria zizanoides Gramineae Root India Lavender Lamiaceae Leaves France Melissa Melissa ofcinalis Lamiaceae Leaves France

Peppermint arvensis Lamiaceae Leaves America

Basil Ocimum basilicum Lamiaceae Leaves Italy

Marjoram Origanum majorana Lamiaceae Flower Egypt

Patchouli Pogostemon cablin Lamiaceae Leaves Malaysia

Rosemary Rosmarinus ofcinalis Lamiaceae Leaves Morocco Clary sage Salvia sclarea Lamiaceae Leaves Russia

Litsea Litsea cubeba Fruit China

Ravensara Ravensara aromatic Lauraceae Leaves Madagascar

Eucalypyus Eucalyptus globulus Myrtaceae Leaves Australia

Tea tree Melaleuca alternifolia Myrtaceae Leaves Australia

Cajeput Melaleuca leucadendra Myrtaceae Leaves Australia

Niaouli Melaleuca viridifora Myrtaceae Leaves Australia Cedarwood Cedrus atlantica Pinaceae Bark America

Lignum cedrium Cedrus deodara Pinaceae Heart wood America

Black pepper Piper nigrum Piperaceae Fruit India

Bergamot Citrus aurantium bergamia Rutaceae Peel Italy

Neroli Citrus aurantium amara Rutaceae Flower Egypt

Orange Citrus aurantium dulcis Rutaceae Peel Italy Grapefruit Citrus grandis Rutaceae Peel Italy

Page 4/24 oil source of plant family name part origin Lemon Citrus limon Rutaceae Peel Italy Mandarin Citrus reticulate Rutaceae Peel Italy Seed Anethum graveolens Umbelliferae Seed China Coriandrum sativum Umbelliferae Fruit China Carum carvi Umbelliferae Seed China Foeniculum vulgre Umbelliferae Seeds Hungary Chuanqiong Ligusticum chuanxiong Umbelliferae Root China Verbena Verbena ofcinalis Verbenaceaex Leaves Spain Zingiber ofcinale Zingiberaceae Stem China 2.3. GC-MS

The chemical analysis of EOs compounds was determined by GC-MS using an Agilent 6890A/5975C, equipped with a HP-5 capillary column. Analytical conditions were as follows: The GC oven temperature was set at 50°C for 10 min, and raised to 280°C at 10°C/min; He was the carrier gas at 1.0 ml/min; the injection of 1.0 µl; and split ratio of 1:50. The chemical components were identifed by NIST mass spectrometry Library (NIST 11.0) and retention index (RI). The relative indices were determined in relation to the series of n-alkanes, with respect to those reported in the literature (Adams, 2007). 2.4. Antifungal assay

The antifungal activity of EOs and active compounds were examined using an in vitro assay by Xie et al. (2017a). Briefy, 25–400 µg/ml of EOs or their major constituent were added to 20 ml sterilize PDA medium and poured into in 9 cm petri dishes, then inoculation mycelial disc (5 mm) were pace in the center of each dish and incubated at 26 ± 1°C for 5–7 days. Three replicates were done for each dose. When the mycelia reached the edge of control plates (only distilled water), antifungal indices were calculated. 2.5 Membrane integrity determination 2.5.1 Effect of EOs on fungal membrane integrity with propidium iodide (PI) dyeing

Membrane integrity of T. hirsuta and L. sulphureus was determined following Yan et al. (2020) method with a confocal laser scanning microscope (CLSM). The fungi were incubated for 24 h in PDB containing the four EOs and its major constituents (1 µl/ml), respectively, and then collected mycelia and stained with PI (1 µg/ml) for 30 min at 28°C in dark. After staining, the mycelia were washed three times with the phosphate buffered saline (PBS, PH 7.4) to remove residual dye. Then use CLSM (Zeiss LSM880,

Page 5/24 Germany) to observe PI with excitation/emission wavelengths 561 nm/591 to 635 nm. Each experiment was repeated three times. 2.5.2 Effect of EOs on leakage of fungal nucleic acid and protein

Fungal nucleic acid and protein leakage were measured according to the methods of Shao et al. (2013) with slight modifcations. Fungi were incubated for 24 h in PDB with four EOs or their major constituent (1 µl/ml), then supernatant was used for nucleic acid and protein leakage determination, which was quantifed using NanoDrop ONE (Thermo SCIENTIFIC). The experiment was performed in three replicates each. 2.5.3 Effect of EOs on fungal soluble sugar content

Soluble sugar content was measured according to the anthrone-sulfuric acid method by Smith et al. (1985), with slight modifcations. 800 µl of fltrates and 100 µl of anthrone-ethyl acetate were mixed, then

1 ml of H2SO4 was added and incubated at 95°C for 10 min, and cooled down at room temperature. Finally, the absorbance was recorded at 620 nm. 2.6. Statistical analyses

The experiments were performed in three replicates, and the experimental results were obtained from mean ± SD. The data of inhibition rate was analyzed using one-way ANOVA by Duncan’s test (p < 0.05).

3. Results 3.1. Antifungal activity of the EOs

The antifungal activity of 37 plant EOs against two wood-decay fungi (Table 2), of these, 4 EOs, litsea (Litsea cubeba), melissa (Melissa ofcinalis), palmarosa (Cymbopogon martini), and verbena (Verbena ofcinalis), achieved 100% inhibition of T. hirsuta and L. sulphureus at 400 µg/ml. The litsea and verbena EOs showed 100% antifungal activity when the concentration was decreased to 200 µg/ml (Fig. 1A-D).

Page 6/24 Table 2 Antifungal activities of essential oils against white-rot fungs T. hirsuta and brown-rot fungs L. sulphureus Plant species Inhibition (%, mean ± SD)

T. hirsuta L. sulphureus

Chamomile 62.2 ± 1.3 bc 77.0 ± 1.6 b Wormwood 4.1 ± 2.0 jk 8.1 ± 1.0 jk Cypress 21.5 ± 1.5 fghij 23.3 ± 1.3 fghij Juniper berry 25.6 ± 1.3 fghi 26.3 ± 0.4 fghij Palmarosa 100 a 100 a Citronella 51.5 ± 1.6 cd 75.2 ± 2.6 b

Vetiver 76.7 ± 2.8 b 83.0 ± 1.3 ab

Lavender 0 k 21.1 ± 2.3 ghij

Melissa 100 a 100 a

Peppermint 23.0 ± 2.0 fghij 42.2 ± 3.4 def

Basil 64.8 ± 1.3 bc 100 a

Marjoram 28.5 ± 8.1 efghi 19.6 ± 0.7 ghij

Patchouli 68.9 ± 2.3 bc 83.3 ± 2.3 ab

Rosemary 23.0 ± 2.0 fghij 34.4 ± 1.3 efg

Clary sage 26.3 ± 1.6 fghi 23.7 ± 1.6 fghij

Litsea 100 a 100 a Ravensara 38.1 ± 1.0 def 18.1 ± 1.6 ghijk

Eucalypyus 14.1 ± 2.3 hijk 0 k

Tea tree 15.2 ± 1.6 ghijk 10.0 ± 2.2 jk

Cajeput 15.2 ± 1.6 ghijk 18.1 ± 3.5 ghijk

Niaouli 25.9 ± 0.7 fghi 26.3 ± 2.6 fghij

Cedarwood 34.4 ± 1.3 defgh 37.4 ± 1.6 defg

Lignum cedrium 26.7 ± 1.7 fghi 13.0 ± 1.6 hijk

Black pepper 26.7 ± 1.3 fghi 53.7 ± 3.2 cde

Bergamot 0 k 26.0 ± 1.6 fghij

400 µg/ml was treated; Means within a column followed by the same letters are not signifcantly different (P < 0.05, Duncan’s test).

Page 7/24 Plant species Inhibition (%, mean ± SD)

T. hirsuta L. sulphureus Neroli 17.0 ± 2.0 fghijk 31.9 ± 1.6 fgh Orange 0 k 11.9 ± 2.3 ijk Grapefruit 0 k 0 k Lemon 0 k 18.1 ± 1.3 ghijk Mandarin 13.0 ± 1.3 ijk 7.8 ± 0.6 jk Dill Seed 29.3 ± 2.7 efghi 30.0 ± 2.3 fghi Coriander 49.6 ± 1.3 cde 53.7 ± 3.2 cde Caraway 60.7 ± 1.3 bc 100 a

Fennel 55.6 ± 1.7 bcd 71.1 ± 1.9 bc

Chuanqiong 35.9 ± 2.1 defg 54.4 ± 1.3 cd

Verbena 100 a 100 a

Ginger 3.7 ± 1.3 jk 34.8 ± 2.9 efg

400 µg/ml was treated; Means within a column followed by the same letters are not signifcantly different (P < 0.05, Duncan’s test).

The antifungal activity of 4 EOs against two wood-decay fungi was given in Table 3. The IC50 values of litsea, verbena, palmarosa, and melissa on T. hirsuta were 72.3, 79.8, 154.1, and 156.3 µg/ml, respectively. In addition, their IC50 values of against L. sulphureus were 40.2, 59.0, 142.0, and 143.2 µg/ml (Table 3), respectively.

Page 8/24 Table 3 IC50 values (µg/ml) of the essential oils and major component against wood-rot fungus T. hirsuta and L. sulphureus Essential oils T. hirsuta L. sulphureus

a 2b IC (CI ) 2 IC50 (CI95) χ 50 95 χ

litsea 72.3 (60.1–87.3) 3.549 40.2 (28.7–51.6) 2.914 melissa 156.3 (127.3-196.5) 9.953 143.2 (116.8-178.7) 6.856 palmarosa 154.1 (126.0-192.5) 8.704 142.0 (117.2-174.2) 5.597 verbena 79.8 (67.1–95.3) 3.471 59.0 (46.5–73.6) 5.656 geranial 56.6 (44.8–70.0) 3.954 33.3 (21.5–44.0) 2.554

geraniol 99.2 (77.4-128.2) 5.728 64.0 (50.4–80.0) 8.840

neral 66.3 (53.6–81.6) 5.077 40.6 (29.0-52.1) 2.885

citral 57.7 (45.3–71.9) 5.062 40.0 (28.6–51.2) 3.013

a Value in µg/ml and CI95-95% confdence intervals, compounds activity is considered signifcantly different when the 95% CI fail to overlap.

b Pearson χ2statistic with P values indicating goodness-of-ft for data to the expected probit response mode 3.2 Chemical compositions of the EOs

The chemical compositions of litsea, melissa, palmarosa, and verbena EOs were shown in Table 4. The major constituents of litsea oil were geranial (32.12%), neral (29.43%), limonene (16.99%), linalool (2.18%), and myrcene (2.17%). The main components in melissa oil were geraniol (31.0%), followed by citronellal (20.84%), citronellol (12.87%), elemol (6.22%), and β-elemene (3.99%). Geraniol (77.42%) was the most abundant, followed by geranyl acetate (12.29%), linalool (3.36%), caryophyllene (2.07%), and nerol (2.02%), in palmarosa oil. The rich component in verbena oil was citral (42.57%), followed by neral (37.32%), geranyl acetate (3.54%), geraniol (3.41%), and linalool (1.37%).

Page 9/24 Table 4 Chemical composition of the 4 essential oils assayed for fungicidal activity No Components RIa LIT RIb litsea melissa palmarosa verbena

1 α-Pinene 939 932 1.97 3.78 0.59 1.10 2 Camphene 954 946 0.47 1.11 3.36 0.22 3 β-Pinene 979 974 1.35 1.09 2.02 0.96 4 Myrcene 991 988 2.17 20.84 77.42 1.37 5 3-Carene 1013 1008 16.99 12.87 0.58 0.43 6 Limonene 1027 1024 0.56 0.33 1.23 0.55 7 β-Phellandrene 1028 1025 2.18 31.00 12.29 0.52

8 1,8-Cineole 1034 1026 1.74 3.46 0.44 37.32

9 β-Ocimene 1038 1032 0.68 0.81 2.07 3.41

10 Linalool 1097 1095 1.03 3.88 100 42.57

11 Isopulegol 1141 1144 0.69 1.66 0.59 3.54

12 Citronellal 1154 1148 1.58 3.99 97.34 0.13

13 Terpinen-4-ol 1177 1174 29.43 1.03 2.07 0.77

14 α-Terpineol 1191 1186 0.90 0.50 0.10

15 Nerol 1228 1227 0.20 2.28 0.18

16 Citronellol 1233 1223 32.12 1.32 0.29

17 Pulegone 1235 1233 0.25 3.84 93.46 18 Neral 1240 1235 0.23 6.22 1.32

19 Geraniol 1250 1249 1.11 100 90.8

20 Anethole 1254 1254 0.11 3.78 1.05

21 Geranial 1272 1264 0.25 77.05 0.29

22 Citronellyl formate 1277 1271 96.01 12.96

23 Citral 1316 1302 23.51 6.22

24 α-Cubebene 1345 1345 70.55

25 Geranyl acetate 1352 1350 1.70 a RI, linear retention indices on HP-5MS column, experimen0t.a2l5ly determined using homologue series of n-alkanes. b Relative retention indices taken from Adams.

Page 10/24 No Components RIa LIT RIb litsea melissa palmarosa verbena 26 Eugenol 1356 1355 27 Neryl acetate 1365 1359 28 β-Elemene 1391 1389 29 Caryophyllene 1419 1417 30 α-Humulene 1455 1452 31 α-Amorphene 1473 1483 32 γ-Muurolene 1480 1476 33 Germacrene D 1485 1484 34 α-Muurolene 1500 1500

35 δ-Cadinene 1523 1522

36 Elemol 1549 1548

37 Caryophyllene oxide 1587 1582

Total identifed (%)

Monoterpene hydrocarbons

Oxygenated monoterpenes

Sesquiterpene hydrocarbons

Oxygenated sesquiterpenes

a RI, linear retention indices on HP-5MS column, experimentally determined using homologue series of n-alkanes.

b Relative retention indices taken from Adams. 3.3 Antifungal activity of the major constituents

To further research the relationship between EOs and it major constituents and antifungal activity, geranial, geraniol, neral, and citral, which were the major constituents of the litsea, melissa, palmarosa, and verbena EO, respectively, were selected for this study. Figure 2 showed geranial, geraniol, neral, and citral completely inhibited T. hirsuta growth at 400 µg/ml. When the concentration was decreased to 200 µg/ml, geranial, neral, and citral caused complete inhibition (Fig. 2). When the concentration of the constituent was 200 µg/ml, geranial, geraniol, neral, and citral completely inhibited the growth of L. sulphureus (Fig. 2).

As shown in Table 3, geranial and citral exhibited the best antifungal activities against T. hirsuta, with

IC50 values of 56.6 and 57.7 µg/ml, respectively. In addition, the IC50 values of geranial, citral, neral, and

Page 11/24 geraniol against L. sulphureus were 33.3, 40.0, 40.6, and 64.0 µg/ml, respectively (Table 3).

3.4 Membrane integrity of T. hirsuta and L. sulphureus exposed to 4 EOs and their major constituents

PI can detect the membrane permeability, which enters the damaged plasma membranes and combines with nucleic acids to produce red fuorescence. PI staining was used to determine whether four EOs (litsea, melissa, palmarosa, and verbena) and their major constituents (geranial, geraniol, neral, and citral, respectively) led to damage of membrane permeability in T. hirsuta and L. sulphureus. Four EOs and their major constituents were used to treat mycelium and stained by PI, however, the control mycelium were not stained (Fig. 3A, B), indicating a considerable destruction in membrane integrity.

To confrm that the four tested EOs and their major constituents could disrupt the cell membrane integrity of T. hirsuta and L. sulphureus, the leakage of intracellular content was determined. The results are represented in Fig. 4, after being treated with the four EOs and major constituents (1 µl/ml) for 24 h, the nucleotides, proteins, and soluble sugars in suspensions of T. hirsuta and L. sulphureus were released in a superior signifcantly quantity than that in the control mycelia. Exposure to litsea, melissa, palmarosa, and verbena EOs, the release of nucleotides, proteins, and soluble sugars signifcantly increased (Fig. 4A- C), with litsea exhibiting the strongest impact. Similar results were obtained for the major constituents, geranial, geraniol, neral, and citral for the leakage of cellular components (Fig. 4A-C), with geranial exhibiting the strongest impact. In addition, the four EOs and their major constituents caused leakage of nucleotides, proteins, and soluble sugars in L. sulphureus than T. hirsuta.

4. Discussion

It is generally known that the fungicidal, insecticidal, and nematocidal activities of plant EOs can be attributed to various compounds, notably alcohols, aldehydes, phenol, terpenes, and terpenoids (Boulogne et al. 2012; Tak and Isman 2017; Benelli et al. 2018; Liu et al. 2019; Gong and Ren 2020). In previous studies, the chemical analysis of litsea, melissa, palmarosa, and verbena has been reported (Seo et al. 2009; De Martino et al. 2011; Si et al. 2012; Kakaraparthi et al. 2015; Rehman et al. 2017; Khalili et al. 2018; Kittler et al. 2018a,b; Pouyanfar et al. 2018). Comparisons between previous results revealed differences in the ratios of major and minor constituents. Xie et al. (2012) and Kakaraparthi et al. (2015) found that the chemical composition of EOs differ widely with genotype, cultivation and production conditions, environment factors, and extraction methods.

In this study, litsea, melissa, palmarosa, and verbena EOs had excellent antifungal activity against wood- rotting fungi, which had not been reported previously. In our previous study we had showed that, C. citratus, C. zeylanicum, and O. vulgare EOs had good antifungal activity against T. hirsuta (IC50 = 79.1–

96.9 µg/ml) and L. sulphureus (IC50 = 36.9–69.2 µg/ml) (Xie et al. 2017a). Similarly, Cryptomeria japonica heartwood EO (IC50, 39 µg/ml) and C. japonica sapwood (IC50, 94 µg/ml) exhibited to have far more strong antifungal effect against L. sulphureus (Cheng et al. 2005). In another investigation, Wang et al. (2005) and Cheng et al. (2006) demonstrated that the growth was completely inhibited by 200 µg/ml

Page 12/24 C. osmophloeum EO against L. sulphureus. On the other hand, the extract of C. konishii (IC50, 62 µg/ml) showed excellent antifungal activity (Cheng et al. 2012). These results demonstrated that litsea, melissa, palmarosa, and verbena EOs have excellent antifungal activity.

To evaluate the relationship between the main constituents and antifungal activity, 4 major constituents of these 4 EOs were selected and tested their antifungal activity. In our study, geranial, geraniol, neral, and citral, which were the main constituents of litsea, melissa, palmarosa, and verbena EOs, respectively, exhibited excellent antifungal activity. Similar results showed that eugenol is the major agent responsible for the strong antifungal activity of oil (Cheng et al. 2008; Komala et al. 2012; Matan et al. 2014; Xie et al. 2017a). In addition, our previous studies showed that six EOs exhibited antifungal activity, which contributed to their major constituent (Xie et al. 2017a). Similarly, in previous studies, Carum capticum oil exhibited strong toxicity contributed by thymol (Singh et al. 2004; Park et al. 2007). These results demonstrate that EOs have excellent antifungal activity contributed by their major constituents.

The structure-activity relationships (SARs) of EO monoterpenoids against fungi have been well studied (Zhang et al. 2016a; Xie et al. 2017a, b). The SAR of monoterpenoids and antifungal activity against decay fungi was investigated by Xie et al. (2017a). They found that aldehyde compounds (cinnamaldehyde and citral) generally had more antifungal activity to wood-decay fungi than alcohols (citronellol). In our study, aldehyde compounds (neral, citral, and geranial) exhibited stronger antifungal activity than the alcohol compound (geraniol). Similarly, Zhang et al. (2016a) reported that the aldehyde compound (citral) demonstrated higher activity than alcohol compounds (β-citronellol, geraniol, and 3, 7- dimethyl-1-octanol) against wood-decay fungi. These results demonstrated that α, β-unsaturated carbonyl compounds (neral, citral, and geranial) had a stronger active antifungal effect. Moreover, previous studies reported that aldehydes exhibited the strongest antitermitic activity (Xie et al. 2014). In another investigation, α, β-unsaturated carbonyl compounds were important in insecticidal, fungicidal, and nematocidal activities (Kim et al. 2008; Lee et al. 2008; Seo et al. 2009). This might indicate that the double bond at the α, β position in carbonyl compounds enhances insecticidal, fungicidal, and nematocidal activities.

In this study, the litsea, melissa, palmarosa, and verbena EOs and their major constituents (geranial, geraniol, neral, and citral) had excellent antifungal activity against the two tested fungi. In addition, some researchers have found that these oils and their major constituents have excellent termiticidal properties. Seo et al. (2009) reported that litsea EO had fumigant antitermitic activity against the Japanese termite (Reticulitermes speratus). Similarly, the EO from palmarosa exhibited good antitermitic activity against Nasutitermes corniger (Lima et al. 2013). In our previous study, citral and geraniol exhibited excellent antitermitic activity (Xie et al. 2014). Similarly, geranial, geraniol, and neral also have been demonstrated excellent termiticidal activity (Seo et al. 2009). Therefore, litsea, melissa, palmarosa, and verbena EOs and their major constituents, geranial, geraniol, neral, and citral, respectively, have promising potential as eco-friendly preservatives.

Page 13/24 Cell membrane permeability is critical to the survival of fungal cells, where the damage of membrane could lead to the outfow of intracellular constituents to result in their death (Zhou et al. 2017; Souza et al. 2020; Yan et al. 2020). In this study, the effects of litsea, melissa, palmarosa, and verbena EOs on the integrity of mycelium membranes of T. hirsuta and L. sulphureus were observed by confocal microscopy. The PI staining results demonstrated that litsea, melissa, palmarosa, and verbena EOs disrupted the membrane integrity, with litsea having the highest effect. Similar results have been previously reported for Fusarium solani conidia treated with Aniba canelilla and Aniba parvifora EOs, indicating damage to the conidia’s cytoplasmic membrane (Souza et al. 2020). Yan et al. (2020) also demonstrated that Mentha spicata, M. piperita, and Thymus vulgaris (CT carvacrol and CT thymol) EOs inhibited Rhizopus stolonifer growth by destroying the permeability of the cell membrane. In general, EO had an effective antifungal activity attributed to its major constituent (Xie et al. 2017a). This study found that geranial, geraniol, neral, and citral disrupted the cell membrane integrity of T. hirsuta and L. sulphureus hyphae. Similarly, Tian et al. (2015), Yun and Lee (2017), and Li et al. (2018) reported that ethyl p-coumarate, perillaldehyde, and silymarin had high antifungal activity, which can be attributed to their destruction of the permeability of the fungal plasma membrane. Kalily et al. (2016) demonstrated that linaool destroyed the permeability of the cell membrane, resulting in leakage of intracellular components and cell death. Zhang et al. (2016b) and Zhou et al. (2017) also reported that carvacrol, cinnamaldehyde, and eugenol could disrupt the plasma membrane of Escherichia coli and R. stolonifera, inducing the intracellular contents leakage.

In this study, litsea, melissa, palmarosa, and verbena EOs and their major constituents, geranial, geraniol neral, and citral, respectively, resulted in the leakage of cytoplasmic contents of nuclei acids, proteins, and sugars increased signifcantly, indicating the breakdown of plasma membrane structures and function. This is consistent with PI staining results. Similarly, Souza et al. (2020) and Yan et al. (2020) showed that A. canelilla, A. parvifora, and T. vulgaris EOs damaged the plasma membrane of R. stolonifer and F. solani and resulted in the leakage of intercellular electrolyte. Zhou et al. (2017) also demonstrated that carvacrol and eugenol could result in the damage of membrane permeability, causing the outfow of cytoplasm, nucleic acid, and protein content of R. stolonifer.

5. Conclusion

The present work reported the antifungal properties and mechanism of the essential oils from Litsea (L. cubeba), Melissa (M. ofcinalis), Palmarosa (C. martini) and Verbena (V. ofcinalis) and their major active constituents. The 4 EOs and their major active constituents signifcantly inhibited mycelial growth of T. hirsuta and L. sulphureus through disrupting plasma membrane integrity, and resulting in leakage of nucleic acid, protein, and soluble sugar. The essential oils of litsea, melissa, palmarosa and verbena and their major compounds have potential as environmental-friendly fungicides.

Declarations

Ethical approval

Page 14/24 Not applicable.

Consent to participate

Not applicable.

Consent to publish

All authors whose names appear on the submission approved the version to be published and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Authors Contributions

Yongjian Xie, Xi Yang, Hui Han, Zhilin Zhang and Dayu Zhang carried out the experimental stages, manuscript preparation, and statistical analysis.

Funding

This work was supported by Zhejiang A&F University research fund for fnancial supports (No. 2012FR087, 2014FR009).

Competing interests

The authors declare no competing interests.

Availability of data and materials

Not applicable.

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Figures

Page 20/24 Figure 1

Antifungal activity of the selected essential oils (A: litsea; B: melissa; C: palmarosa; D: verbena) against wood-rot fungus T. hirsuta and L. sulphureus. Each experiment was performed × 3 and the data averaged (n = 3). Numbers followed by different letters (a-g) are signifcantly different at level of P < 0.05 according to Duncan’s test.

Page 21/24 Figure 2

Antifungal activity of major component of the selected essential oils (A: geranial; B: geraniol; C: neral; D: citral) against wood-rot fungus T. hirsuta and L. sulphureus. Each experiment was performed × 3 and the data averaged (n = 3). Numbers followed by different letters (a-g) are signifcantly different at level of P < 0.05 according to Duncan’s test.

Page 22/24 Figure 3

Confocal laser scanning microscopy images of T. hirsuta (A) and L. sulphureus (B) mycelium membrane integrity exposed to 4 essential oils and their major components at 1 μl/ml. DIC: differential interference contrast images without fuorescence. FL: red fuorescence images with propidiumiodide (PI) combined with nucleic acid. Bar = 50.0 μm.

Page 23/24 Figure 4

Effects of 4 essential oils and their major components at 1 μl/ml on leakage of nucleic acid (A), protein (B), and soluble sugar (C) of T. hirsuta and L. sulphureus mycelium. Each value is the mean for three replicates, and means with different letters are signifcantly different based on Duncan’s (P < 0.05).

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